Data from Class I β-tubulin mutations in 2-methoxyestradiol-resistant acute lymphoblastic leukemia cells: implications for drug-target interactions

Abstract

<div>Abstract<p>2-Methoxyestradiol (2ME2) is a naturally occurring derivative of estradiol that has been shown to be an active small molecule that has antitumor and antiangiogenic properties. 2ME2 binds to β-tubulin near the colchicine-binding site, inhibits microtubule polymerization, and induces mitotic arrest. To improve understanding of the mechanisms of action and resistance to 2ME2, we selected leukemia cells, CCRF-CEM, that display increasing resistance to 2ME2, and three of the highly resistant sublines were chosen for detailed analysis. The 2ME2 cells selected in 7.2 to 28.8 μmol/L were found to be 47- to 107-fold resistant to 2ME2 and exhibited low levels of cross-resistance to vinblastine. Two of the lowest 2ME2-resistant sublines were significantly hypersensitive to colchicine and epothilone B, but the hypersensitive effects were lost in the highest 2ME2-resistant subline. Moreover, 2ME2-resistant cells require 10-fold higher concentrations of 2ME2 to induce G<sub>2</sub>-M cell cycle arrest and have higher amounts of tubulin polymer compared with parental cells. Gene and protein sequencing revealed four class I β-tubulin mutations, S25N, D197N, A248T, and K350N, in the 2ME2-resistant cells. The S25N mutation is within the paclitaxel-binding site, whereas A248T and K350N are within the colchicine-binding site on β-tubulin, yet the resistant cells were not cross-resistant to paclitaxel or colchicine. This strongly suggests that the mutations have induced conformational changes to the binding site that resulted in 2ME2 resistance. The 2ME2-resistant leukemia cells provide novel insights into microtubule stability and drug-target interactions. [Mol Cancer Ther 2008;7(9):3150–9]</p></div>